Methods for disrupting biofilms

ABSTRACT

The present invention provides methods for disrupting biofilms and/or preventing the formation of biofilms along with medical items that contain a coating or covering made of compositions for achieving such disruption or prevention. In particular, the compositions of the present invention provide random-sequence peptide mixtures for use in disrupting bacterial biofilms; the random-sequence peptides having hydrophobic and/or cationic amino acids, wherein the ratio of the total hydrophobic and cationic amino acids in the mixture is predefined.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 16/074,895filed Aug. 2, 2018, now U.S. Pat. No. 11,XXX,XXX, which is the USNational Stage Filing of PCT/IL2017/050122 filed Feb. 2, 2017, whichclaims priority from U.S. provisional application No. 62/291,117 filedFeb. 4, 2016.

FIELD OF THE INVENTION

The present invention relates to methods for disrupting biofilms. Inparticular, the present invention relates to random-sequence syntheticpeptide mixtures for use in disrupting biofilms and/or preventing theformation of biofilms.

BACKGROUND OF THE INVENTION

Biofilm is an assemblage of surface-associated microbial cells that areenclosed in an extracellular polymeric substance matrix. Biofilms beingformed when bacteria adhere to surfaces in aqueous environments andbegin to excrete a slimy, glue-like substance that can anchor them toall kinds of materials, such as metals, plastics, soil particles,medical implant materials and, most significantly, human or animaltissues. Biofilms are composed primarily of microbial cells andextracellular polymeric substance (EPS) matrix. EPS may account for 50%to 90% of the total organic carbon of biofilms and can be considered theprimary matrix material of the biofilm. EPS may vary in chemical andphysical properties, but it is primarily composed of polysaccharides.Additional biofilm substances are excreted proteins and nucleic acids.The extracellular matrix protects the microorganisms embedded within thebiofilm and facilitates the communication between microorganisms throughbiochemical signals.

Clinical and public health microbiologists' recognition that microbialbiofilms are ubiquitous in nature has resulted in the study of a numberof infectious diseases from a biofilm perspective. For example, cysticfibrosis, native valve endocarditis, otitis media, periodontitis, andchronic prostatitis, are all appear to be caused by biofilm-associatedmicroorganisms. In addition, a spectrum of indwelling medical devices orother devices used in the health-care environment have been shown toharbor biofilms, resulting in measurable rates of device-associatedinfections.

Staphylococcus aureus (S. aureus) is one of the most seriousbiofilm-forming pathogens that cause complications ranging from minor tolife-threatening infections. Methicillin-resistant S. aureus (MRSA)isolated from clinical environments has a high probability of formingbiofilms. MRSA can cause a variety of problems ranging from skininfections to pneumonia, bloodstream infections, and sepsis. MRSAbiofilm life cycle is believed to occur in four stages: the initialattachment of bacterial cells to a surface, formation of micro-colonieson the surface of interest, maturation of the micro-colonies into anestablished biofilm, and dispersal of the bacteria from the biofilm.

Biofilm in Food Industry

The demand for minimally processed, easily prepared and ready-to-eat‘fresh’ food products poses major challenges for food safety andquality. Commonly, food contamination leads to spoilage and growth ofpathogenic microorganisms. Contamination of food is common duringslaughtering, processing, packaging and shipping stages of foodprocessing. As food production becomes increasingly automated, thenumber of surfaces with which foods come into contact, and the potentialfor cross-contamination, increases. These surfaces are highly sensitiveto microbial attachment and biofilm formation. It is imperative to foodmanufactures to design new approaches that inhibit microbial growth infood by inhibiting and eradicating biofilm formation while maintainingquality, freshness, and safety.

Host-Defense Peptides

Several Host-defense peptides (HDPs) were reported to prevent biofilmformation but only few were able to target existing mature biofilms.HDPs are produced by eukaryotes as part of the innate immune response tobacterial infection. The anti-microbial mechanism of action of HDP isattributed mainly to the disruption of bacterial membranes. They oftendisplay a characteristic selectivity, favoring an attack on prokaryoticmembranes relative to eukaryotic membranes. HDPs are rich in hydrophobicresidues which mediate disruptive interactions with the hydrophobicinterior of the bacterial membrane lipid bilayer. The broad moleculardiversity among HDPs suggests that their activity and selectivity is nottightly coupled to specific features of amino acid sequence or peptideconformation. This situation has encouraged the design of several typesof random cationic polymers that mimic HDPs activity by displaying broadantimicrobial activity. Grinstaff and his co-workers showed thatpoly-amido-saccharide (PAS) prepared by a controlled anionicpolymerization of β-lactam monomers derived from either glucose orgalactose can inhibit biofilm formation (Dane et al., Chem Sci 2014, 5).Interestingly, it was shown previously that D-Amino acids, such asD-phenylalanine and D-tyrosine prevented biofilm development inStaphylococcus aureus and Bacillus subtilis.

Recently, a method to generate mixtures of random cationic peptides wasdeveloped by the inventor of the present invention and co-workers(Hayouka et al., J. Am Chem Soc. 2013, 135: 32). A method of solid-phasepeptide synthesis was employed using a mixture of two amino acids ineach elongation step in a defined proportion. Using this method,synthesis of copolymers via n coupling steps generated 2^(n) sequencesof random peptides with a defined composition and controlled chainlength (Hayouka et al., ibid).

U.S. Pat. No. 6,172,038 discloses non-natural synthetic peptidescomprising both L- and D-amino acid residues designated diastereomericpeptides with a net positive charge that is greater than +1. Somesynthetic peptides consist of at least one hydrophobic amino acid and atleast one positively charged amino acid, in which at least one of theamino acid residues is a D-amino acid.

U.S. Pat. No. 9,499,594 discloses methods for the prevention, control,disruption and treatment of bacterial biofilms with lysine, particularlylysine having capability to kill Staphylococcal bacteria, including drugresistant Staphylococcus aureus.

The inventor of the present invention and co-workers disclose thatrandom-sequence peptide mixtures are capable of controlling and managingMRSA biofilms (Chem. Commun. April 2016, 52: 7102-7105).

The development of biofilms that allow for an aggregated cell bacteriato be increasingly antibiotic resistant as well as the generalincreasing resistance of bacteria to antimicrobial substances,necessitate the development of new methods for destroying and preventingbiofilms.

Thus, there remains an unmet need for improved methods of preventing theformation of biofilm and/or eradicating biofilms.

SUMMARY OF THE INVENTION

The present invention provides methods for disrupting and/or preventingthe formation of biofilms. In particular, the present invention providesmethods for disrupting biofilms by applying mixtures comprisingrandom-sequence peptides to the biofilm. The mixtures comprisehydrophobic and cationic amino acids. The length of the random-sequencepeptides as well as the ratio of the total hydrophobic to cationic aminoacids in the mixture is highly controlled and pre-defined.

The present invention is based in part on the unexpected findings thatrandom-sequence peptide mixtures comprising hydrophobic and cationicamino acids, when contacted with a mature biofilm ofantibiotic-resistant bacteria, were capable of disrupting the biofilm ofthe antibiotic-resistant bacteria as well as of killing these bacteria.The anti-biofilm activity of the random-sequence peptide mixtures wassimilar to the anti-biofilm activity of daptomycin, a lipopeptideantibiotic agent that is being used in the treatment of systemic andlife-threatening infections caused by MRSA.

The negatively charged exopolysaccharides which form the biofilms arevery effective in protecting microbial cells from positively chargedantibacterial agents by restricting their permeation, possibly throughbinding. Unexpectedly, the random sequence peptide mixtures of thepresent invention which comprise cationic amino acids were found topenetrate an existing biofilm, disrupt its structure, and kill itsembedded cells.

It is now disclosed that mixtures of homochiral or heterochiral 20-merrandom-sequence peptides of phenylalanine and lysine (^(L)F^(L)K or^(L)F^(D)K) at a 1:1 ratio prevented biofilm formations as well asdisrupted mature bacterial biofilm. The heterochiral mixtures showedhigher activity in biofilm disruption as compared to the homochiralmixtures. The homochiral or heterochiral mixtures were also shown to behighly potent in killing bacterial cells embedded within the biofilmbiomass. The killing effect of the heterochiral mixtures was higher thanthat of the homochiral mixtures and was highly selective to bacterialcells because human red blood cells were essentially unaffected by theseheterochiral mixtures. Moreover, the homochiral or heterochiral mixturesof phenylalanine and lysine (^(L)F^(L)K or ^(L)F^(D)K) showed anexceptional low cytotoxicity to human intestinal epithelial cell line,even at high concentrations.

It is further disclosed that the random-sequence peptide mixtures of thepresent invention were capable of penetrating into bacterial cells.Without being bound to any mechanism of action, the capability of therandom-sequence peptides to penetrate into bacterial cells may indicatethat the peptides may cause damage to the bacterial membrane and henceenhance the entry of the peptides into the bacterial cells.Advantageously, the methods of the present invention are not associatedwith risks of acquired resistance as occur when one specific peptidesequence or a single antimicrobial agent are being used. In addition,the synthesis of random peptide mixtures is simple and cost-effectiverelative to the synthesis of specific peptide sequences.

Thus, the methods of disrupting biofilms, and specifically ofantibiotic-resistant bacterial biofilms, of the present invention arehighly efficient, selective, reduce the risk of developing bacterialresistance, and therefore are safe for human use.

According to one aspect, the present invention provides a pharmaceuticalcomposition comprising a mixture of random-sequence peptides for use intreating or preventing a biofilm-associated infection in a subject,wherein the random-sequence peptides are of 3 to 50 amino acid residuesin length, wherein said random-sequence peptides comprise hydrophobicand/or cationic amino acids, and wherein the ratio of the totalhydrophobic and cationic amino acids in the mixture is between 10:1 and1:10.

According to some embodiments, the biofilm-associated infection iscaused by unicellular organisms. According to certain embodiments, theunicellular organism is selected from the group consisting of bacteria,fungi, yeast, and archaea.

According to some embodiments, the biofilm-associated infection iscaused by gram-positive bacteria, gram-negative bacteria, or antibioticresistant bacteria. According to additional embodiments, the bacteriaare gram-positive bacteria. According to further embodiments, thebacteria are gram-negative bacteria.

According to some embodiments, the biofilm-associated infection iscaused by bacteria selected from the group consisting of Staphylococcus,Bacillus subtilis, Streptococcus, Bacillus cereus, E. coli, Listeria,Salmonella, Pseudomonas, Acinetobacter baumannii, Klebsiella pneumoniae,Burkholderia cenocepacia, Helicobacter pylori, and Enterococcus.According to additional embodiments, the Staphylococcus or Streptococcusbacteria are selected from the group consisting of Staphylococcusaureus, Staphylococcus simulans, Streptococcus suis, Staphylococcusepidermidis, Streptococcus equi, Streptococcus agalactiae (GBS),Streptococcus pyogenes (GAS), Streptococcus sanguinis, Streptococcusgordonii, Streptococcus dysgalactiae, Group G Streptococcus, Group EStreptococcus, and Streptococcus pneumonia. Each possibility representsa separate embodiment of the invention.

According to some embodiments, the antibiotic resistant bacteria areselected from the group consisting of methicillin-resistantStaphylococcus aureus (MRSA), vancomycin resistant Staphylococcus aureus(VRSA), daptomycin-resistant Staphylococcus aureus (DRSA),linezolid-resistant Staphylococcus aureus (LRSA), Lactobacillus-MRS(LMRS), and vancomycin-resistant Enterococci (VRE). Each possibilityrepresents a separate embodiment of the invention. According to specificembodiments, the bacteria are Bacillus subtilis. According to additionalembodiments, the bacteria are Staphylococcus aureus. According toadditional embodiments, the bacteria are MRSA. According to additionalembodiments, the bacteria are Lactobacillus-MRS (LMRS). According tosome embodiments, the bacteria are resistant to more than one antibioticagent.

According to some embodiments, the mixture comprises heterochiralpeptides. According to other embodiments, the mixture consists ofhomochiral peptides. According to some embodiments, the amino acids areall D-amino acids. According to additional embodiments, the amino acidsare all L-amino acids. According to specific embodiments, if thehydrophobic amino acid is in the L-configuration, the cationic aminoacid in the D-configuration. According to other embodiments, if thehydrophobic amino acid is in the D-configuration, the cationic aminoacid in the L-configuration. According to yet further embodiments, thehydrophobic and cationic amino acids are in the L-configuration and/orD-configuration.

According to some embodiments, the hydrophobic amino acid is selectedfrom the group consisting of phenylalanine, tryptophan, leucine, valine,alanine, isoleucine, glycine, and a combination thereof. Eachpossibility represents a separate embodiment of the invention. Accordingto additional embodiments, the hydrophobic amino acid is a hydrophobicaromatic amino acid. According to certain embodiments, the hydrophobicaromatic amino acid is tryptophan or phenylalanine. According to oneexemplary embodiment, the hydrophobic amino acid is phenylalanine.According to another exemplary embodiment, the hydrophobic amino acid istryptophan.

According to some embodiments, the cationic amino acid is selected fromthe group consisting of lysine, arginine, histidine, ornithine, di-aminobutyric acid (Dab), and a combination thereof. Each possibilityrepresents a separate embodiment of the invention. According to anexemplary embodiment, the cationic amino acid is lysine.

According to some embodiments, the random-sequence peptide mixtureconsists of one species of a hydrophobic amino acid and one species of acationic amino acid.

According to some embodiments, the hydrophobic amino acid isphenylalanine and the cationic amino acid is lysine. According to otherembodiments, the hydrophobic amino acid is tryptophan and the cationicamino acid is lysine.

According to some embodiments, the ratio of the total hydrophobic andcationic amino acids in the mixture is about 5:1 to 1:5, alternativelyabout 3:1 to 1:3, and further alternatively about 1:1.

According to some embodiments, the random-sequence peptides are of 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 35amino acid residues in length or any integer in between. Eachpossibility represents a separate embodiment of the invention.

According to some embodiments, the peptides within the mixture haveidentical number of amino acid residues in length, the number ofresidues ranges between 5 and 25, alternatively between 10 and 25,further alternatively between 15 and 25 residues in length. According toa certain embodiment, the peptides consist of 20 amino acid residues inlength.

According to some embodiments, the biofilm-associated infection isselected from the group consisting of skin, oral, dental, genitourinarytract, and airways infections. According to certain embodiments, thebiofilm-associated infection is selected from the group consisting ofwound infection, Legionnaire's disease, middle-ear infection, acne,dental plaque infection, gingivitis, periodontitis, and cystic fibrosis.

According to some embodiments, the composition further comprises anantimicrobial agent. According to certain embodiments, the antimicrobialagent is an antibiotic agent. According to some embodiments, theantibiotic agent is selected from the group consisting of daptomycin,clindamycin, dicloxacillin, minocycline, nafcillin, oxacillin,ramoplanin, rifampin, triclosan, linezolid, penicillin, cephalosporin,and vancomycin. According to specific embodiments, the antibiotic agentis daptomycin.

According to an additional aspect, the present invention provides amethod of treating or preventing a biofilm-associated infection in asubject comprising administering to the subject in need of suchtreatment a pharmaceutical composition comprising a mixture ofrandom-sequence peptides of 3 to 50 amino acid residues in length,wherein the random-sequence peptides comprise hydrophobic and/orcationic amino acids, and wherein the ratio in the mixture of the totalhydrophobic and cationic amino acids is between 10:1 and 1:10; and apharmaceutically acceptable carrier.

According to another aspect, the present invention provides a method forpreventing biofilm formation, the method comprises applying to a surfaceof an item a composition comprising an effective amount of a mixture ofrandom-sequence peptides of 3 to 50 amino acid residues in length, saidpeptides comprise hydrophobic and/or cationic amino acids, wherein theratio in the mixture of the total hydrophobic and cationic amino acidsis between 10:1 and 1:10.

The biofilm and the peptides are as described hereinabove.

According to some embodiments, the item is a medical device, catheter orimplant.

According to some embodiments, the medical device is selected from thegroup consisting of a cardiac rhythm management device (CRMD), aneurostimulator, a pulse generator, a drug pump or infusion device, aphysiological monitoring device, or a textured or smooth breast implant.

According to some embodiments, the composition is immobilized to thesurface of the medical device, catheter or implant.

According to some embodiments, the surface is coated with or covered bythe composition according to the invention.

According to some embodiments, the item is commercial and industrialwater systems.

According to an additional aspect, the present invention provides amethod of inhibiting planktonic growth of antibiotic-resistant bacteria,the method comprises contacting antibiotic-resistant bacteria with acomposition comprising a mixture of random-sequence peptides of 3 to 50amino acid residues in length, wherein the peptides comprise hydrophobicand/or cationic amino acids, and wherein the ratio in the mixture of thetotal hydrophobic and cationic amino acids is between 10:1 and 1:10; anda pharmaceutically acceptable carrier.

The mixture and the peptides are as described hereinabove.

According to some embodiments, the bacteria are methicillin-resistantStaphylococcus aureus (MRSA).

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of random-sequence peptide mixtures on thegrowth of methicillin-resistant Staphylococcus aureus (MRSA). Clinicallyisolated strain of MRSA was treated with random sequence 20-merphenylalanine-lysine peptide mixtures (homochiral ^(L)F^(L)K orheterochiral ^(L)F^(D)K). Growth-inhibitory activities were measuredafter incubation of 6 hr or 24 hr.

FIG. 2 shows the effect of random sequence peptide mixtures on MRSAbiofilm degradation. Random sequence 20-mer phenylalanine-lysine peptidemixtures (homochiral ^(L)F^(L)K or heterochiral ^(L)F^(D)K) orleucine-lysine peptide mixture (LK) were examined for their effect onMRSA biofilm biomass degradation by crystal violet staining. Degradationwas compared to the antibiotic Daptomycin.

FIG. 3 shows bacterial cell viability in MRSA biofilm after treatmentwith phenylalanine-lysine random-sequence peptide mixture (Homochiral^(L)F^(L)K or heterochiral ^(L)F^(D)K) at a concentration of 100 μg/mL.

FIG. 4 shows bacterial cell counting in MRSA biofilm after treatmentwith the random-sequence peptide mixtures (homochiral ^(L)F^(L)K orheterochiral ^(L)F^(D)K). Daptomycin was used as a positive control.

FIGS. 5A, 5B and 5C are scanning electron micrographs showing the effectof random-sequence peptide mixtures (homochiral ^(L)F^(L)K orheterochiral ^(L)F^(D)K) on mature MRSA biofilm. A mature MRSA biofilmgrown on glass disks was treated for 24 hrs with 100 μg/mL ^(L)F^(L)K(FIG. 5B) or with ^(L)F^(D)K (FIG. 5C). As a control, non-treated matureMRSA biofilm is shown (FIG. 5A).

FIGS. 6A and 6B show the effect of homochiral or heterochiral mixturesof random-sequence peptides consisting of tryptophan and lysine (WK,W^(D)K, ^(D)WK, and ^(D)W^(D)K) on Bacillus subtilis biofilm formation.Lactobacillus-MRS (LMRS) and B. subtilis 3610 not treated with thepeptide mixtures were use as control.

FIGS. 7A, 7B and 7C are representative confocal microscopy images ofMRSA cells treated with labeled random-sequence peptide mixtures.Fluorescently labeled random peptide mixture (F1′-^(L)F^(L)K orF1′-^(L)F^(D)K) were incubated for 1 hr with MRSA cells and thenphotographed using confocal microscopy. FIG. 7A shows MRSA cells, nottreated with the peptides. FIGS. 7B and 7C show MRSA cells treated with(F1′-^(L)F^(L)K or F1′-^(L)F^(D)K), respectively.

FIG. 8 shows the hemolytic activity of increasing concentrations of^(L)F^(L)K and ^(L)F^(D)K towards human red blood cells.

FIG. 9 shows the viability of Caco-2 cells (a human intestinalepithelial cell line) in the presence of increasing concentrations of^(L)F^(L)K, ^(L)F^(D)K, and leucine lysine (^(L)L^(L)K).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutical compositions comprisingrandom-sequence synthetic peptide mixtures for use in treating orpreventing biofilm-associated infections and/or for use in disruptingbiofilm and/or for use in killing bacteria embedded in a biofilm and/orfor use in preventing formation of biofilm, in a subject in need of suchtreatment. The present invention further provides random-sequencesynthetic peptide mixtures for use in preventing biofilm formation on asurface of medical devices, on food processing surfaces, or oncommercial and industrial water systems. The mixture comprises randomsequence peptides of 3 to 50 amino acid residues in length, wherein thepeptides comprise hydrophobic and/or cationic amino acids, and whereinthe ratio in the mixture of the total hydrophobic to cationic aminoacids is highly controlled and pre-defined.

Random-sequence peptide mixtures are significantly advantageous comparedto anti-biofilm specific peptide sequences for two reasons: (1) thesynthesis of random-sequence peptide mixtures is easy and cost-effectiverelative to the synthesis of specific peptide sequences; and (2) themixtures contain many peptides with different amino acid sequences, andas such can be considered to be a cocktail of anti-biofilm agents whichreduce or even eliminate the risks of acquired antibiotic resistance inbacteria.

Advantageously, it is now disclosed that mixtures of synthetic randomsequence peptides comprising hydrophobic and cationic amino acids arehighly efficient in preventing biofilm formation, in eradicating anexisting or mature biofilm, and in killing bacteria embedded orassociated with biofilms. The present invention teaches that specificamino acid sequences of antimicrobial peptides are not essential forpreventing biofilm formation, for eradicating an existing biofilm, andfor killing bacteria associated with biofilm.

The inventors of the present invention showed that mixtures of randomsequence peptides of hydrophobic amino acids and cationic amino acids,e.g., phenylalanine-lysine, tryptophan-lysine, and leucine-lysine,showed high anti-biofilm activity. However, higher anti-biofilm activitywas observed with peptide mixtures of hydrophobic aromatic amino acidsand cationic amino acids, e.g., a phenylalanine-lysine mixture, in someexperimental conditions. In addition, heterochiral mixtures ofhydrophobic aromatic amino acids and cationic amino acids showed higheranti-biofilm activity than homochiral mixtures, and such heterochiralmixtures were found to be essentially non-toxic to human cells.

The term “biofilm” refers to a population of microorganisms, such asbacteria, growing on a surface, wherein the bacteria are encased in amatrix generally composed of polysaccharides, proteins and nucleicacids. In this state, bacteria are less susceptible to both phagocytesand antibiotics. The term “biofilm” is further intended to includebiological films that develop and persist at interfaces in aqueousenvironments.

The terms “disrupting biofilm” and “eradicating biofilm” are used hereininterchangeably and are defined as the ability of the mixtures asdefined in the present invention to degrade an existing or maturebiofilm and/or to inhibit, prevent, or reduce the formation of a biofilmin vitro as well as in vivo and/or to kill microorganisms embedded orassociated with biofilms.

The terms “preventing biofilm formation” or “reducing biofilm formation”are used herein interchangeably and refer to the ability of the mixturesdisclosed herein to avert or reduce the formation of a biofilm bymicroorganisms. According to some embodiments, preventing biofilmformation means inhibiting bacterial attachment to a surface.Additionally, or alternatively, preventing biofilm formation meanskilling the biofilm-forming bacteria.

The term “mixture” as used herein refers to at least two differentrandom-sequence peptides as discloses herein, preferably to a pluralityof random-sequence peptides. According to the invention, the mixture ofrandom-sequence peptides comprises or consists of cationic amino acidsand hydrophobic amino acids. The mixture can be mixed with anotheranti-microbial agent.

The term “random-sequence peptide” as used herein refers to a peptide,the amino acid sequence of which is different from the amino acidsequence of at least one peptide in the mixture. According to someembodiments, the mixture can have up to 2^(n) amino acid sequences,wherein n defines the number of coupling steps in the peptide synthesis,if one species of a cationic amino acid residue and one species of ahydrophobic amino acid residue are present both in L-configuration orD-configuration. According to further embodiments, the mixture can haveup to 4^(n) amino acid sequences, wherein n defines the number ofcoupling steps in the peptide synthesis, if one species of a cationicamino acid residue and one species of a hydrophobic amino acid residueare present in L-configuration and D-configuration. Thus, the number ofrandom-sequence peptides in a mixture is dictated by the length of thepeptides synthesized, the various species of amino acids, and theconfiguration of the amino acids.

According to the invention, the random-sequence peptides comprise orconsist of hydrophobic and/or cationic amino acids. According to someembodiments of the invention, one or more peptides in the mixture,preferably 10% or less, such as less than 1% of the peptides in themixture, comprise or consist of hydrophobic amino acids but are devoidof cationic amino acids, one or more peptides in the mixture, preferablyabout 10% or less, such as less than 1% of the peptides in the mixture,comprise or consist of cationic amino acids but are devoid ofhydrophobic amino acids, and at least about 80% of the peptides in themixture, alternatively at least about 90%, 95%, or preferably at leastabout 99% of the peptides in the mixture comprise or consist of acombination of cationic and hydrophobic amino acids. According to acertain embodiment, the peptides of the mixture comprise or consist of acombination of hydrophobic and cationic amino acid residues.

The term a “cationic amino acid” refers to a positively charged aminoacid, also known as a basic amino acid.

The term “ratio” as applied to the amino acids within a mixture refersto the ratio between different classes of amino acids which constitutethe peptides present in the mixture. For example, “a ratio in themixture of the total hydrophobic and cationic amino acids” refers to thenumber of all hydrophobic amino acids present in or constitute thepeptides of the mixture relative to the number of all cationic aminoacids present in or constitute the peptides of the mixture.

The term “stereoisomeric forms” refers to the L- or D-configuration ofamino acids.

Unless otherwise mentioned or indicated, the amino acids are in Lconfiguration.

The term “homochiral” as used herein refers to amino acids having thesame configuration (i.e., L-configuration or D-configuration).

The term “heterochiral” as used herein refers to amino acids havingdifferent configurations, i.e., one is in the L-configuration and theother is in the D-configuration.

The term “heterochiral mixture” as used herein refers to a mixturecomprising random-sequence peptides; at least one of the peptidesconsists of amino acids in the L-configuration and amino acids in theD-configuration. Preferably, a heterochiral mixture comprises aplurality of peptides consisting of amino acids in the L-configurationand amino acids in the D-amino configuration.

The term “homochiral mixture” as used herein refers to a mixturecomprising random-sequence peptides, wherein all the peptides consist ofamino acids in the L-configuration or in the D-configuration.

The singular forms “a”, “an” and “the” include corresponding pluralreferences unless the context clearly dictates otherwise.

As used herein, the term “about” means plus or minus 10% of thenumerical value indicated.

According to some embodiments, disrupting the biofilm is characterizedby reducing the biofilm biomass by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% compared to a non-treated biofilm. Eachpossibility represents a separate embodiment of the invention.

According to some embodiments, the biofilm formation is reduced by atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to anon-treated site. Each possibility represents a separate embodiment ofthe invention.

According to some embodiments, disrupting the biofilm is characterizedby killing at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ofthe bacterial population in the biofilm.

Cationic amino acids as used herein are selected from cationic orpositively charged amino acids known in the art. Positively chargedamino acids include, but are not limited to, lysine, arginine, andhistidine. Hydrophobic amino acids as used herein are selected fromhydrophobic amino acids known in the art. Hydrophobic amino acidsinclude, but are not limited to, phenylalanine, tryptophan, leucine,isoleucine, glycine, alanine, and valine. According to some embodiments,the hydrophobic amino acid is a hydrophobic aromatic amino acid.According to certain embodiments, the hydrophobic aromatic amino acid isphenylalanine or tryptophan. The peptides of the present inventioncomprise L-amino acids, D-amino acids, or a combination thereof. Theamino acids may also be selected from non-protein amino acids, such as,for example, ornithine. The amino acids of the invention can benon-natural amino acids such as, for example, n-methylated amino acids,γ-amino acids, and ring-substituted phenylalanine.

The peptides of the present invention can further comprise additionalamino acid residues, such as polar or uncharged amino acid residues, forexample, asparagine and/or serine, as long as the ratio between thetotal r cationic amino acid residues and the total hydrophobic aminoacid residues in the mixture is maintained between about 10:1 to 1:10,alternatively about 5:1 to 1:5, alternatively about 3:1 to 1:3, andpreferably about 1:1.

According to some embodiments, the random-sequence peptides in themixture comprise or consist of hydrophobic amino acid residues, cationicamino acid residues, or a combination thereof. According to additionalembodiments, the random-sequence peptides comprise or consist of onespecies of a hydrophobic amino acid residue and one species of cationicamino acid residue. According to exemplary embodiments, therandom-sequence peptide mixture consists of phenylalanine and lysine,tryptophan and lysine, or leucine and lysine. According to a certainembodiment, the random-sequence peptide mixture consists ofphenylalanine and lysine, or tryptophan and lysine.

The random-sequence peptide mixtures of the present invention can besynthesized by a solid phase peptide synthesis method as describedherein below. The random-sequence peptide mixtures can also besynthesized by other solid phase peptide synthesis methods, such as thesolid-phase mix-and-split combinatorial synthesis method, as known inthe art.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprising atherapeutically effective amount of random-sequence synthetic peptidemixtures of the invention and a pharmaceutically acceptable carrier ordiluent, for use in disrupting biofilm or for treating or preventingbiofilm-associated infection in a subject.

The term “pharmaceutical composition” as used herein refers to acomposition comprising at least one pharmaceutically active ingredient.

The pharmaceutical compositions of the present invention comprise apharmaceutically acceptable carrier. The term “carrier” refers to adiluent or vehicle with which the therapeutic compound is administered.Carrier(s) are “acceptable” in the sense of being compatible with theother ingredients of the composition and not deleterious to therecipient thereof. Such pharmaceutical carriers can be sterile liquids,such as water; oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like; polyethylene glycols; glycerin; propylene glycol; orother synthetic solvents. For injectable solutions, water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers for injectablesolutions.

The pharmaceutical composition can further comprise a surfactant.According to some embodiments, the surfactant is a nonionic surfactant.Nonionic surfactants include, but are not limited to, sorbitan fattyacid esters, polyoxysorbitan fatty acid esters, polyoxyalkylene higheralcohol ethers, and polyoxyalkylene higher alcohol esters. Thus,nonionic surfactants include polyoxyethylene sorbitol esters such aspolysorbate 80 (TWEEN® 80), polysorbate 60 (TWEEN® 60) and polysorbate20 (TWEEN® 20), Tyloxapol; polyoxyethylene isooctylphenyl ethers such asTriton X-100, polyoxyethylene nonylphenyl ethers such as NP-40,polyoxyethylene dodecyl ethers such as Brij 58, octyl glucoside, andalkyl maltoside such as n-dodecyl-beta-D-maltoside; Poloxamer 4070;Poloxamer 188; and polyoxyl 40 stearate. Each possibility is a separateembodiment of the invention. TWEEN® and Poloxamer surfactants arepreferred because they are FDA approved for human use.

The pharmaceutical compositions of the present invention can beformulated as a liquid. The liquid composition can be stored as is orcan be stored in a frozen state, or in a dried form for laterreconstitution into a liquid form or other form suitable foradministration to a subject.

The compositions may be suitably formulated for subcutaneous,intramuscular, intraperitoneal or intravenous administration andcomprise sterile aqueous solutions, which are preferably isotonic. Suchformulations are typically prepared by dissolving solid activeingredients in water containing physiologically compatible substancessuch as sodium chloride, glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be prepared in unit ormulti-dose containers, for example, sealed ampoules or vials.

The compositions may incorporate a stabilizer, such as for examplepolyethylene glycol, proteins, saccharides (for example, trehalose),amino acids, inorganic acids and admixtures thereof. Stabilizers areused in aqueous solutions at the appropriate concentration and pH. ThepH of the pharmaceutical composition of the present invention isadjusted to be within the range of 5.0-9.0, preferably within the rangeof 6-8.

According to some embodiments, the compositions of the invention may beformulated for oral administration in liquid solutions, emulsions,suspensions, tablets, dragees, capsules, powder, and the like. Thepharmaceutically-acceptable carriers suitable for preparation of suchcompositions are well known in the art. Proteins or peptides that areorally administered need to be protected as to avoid digestion by thegastrointestinal system.

The mixtures of the invention can be coated with enteric coatinglayer(s) as to protect the peptides from digestion. Enteric coatinglayer(s) may be applied using standard coating techniques. The entericcoating materials may be dissolved or dispersed in organic or aqueoussolvents and may include one or more of the following materials:methacrylic acid copolymers, shellac, hydroxypropylmethylcellulosephthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose,carboxymethylethylcellulose, cellulose acetate phthalate or othersuitable enteric coating polymer(s). The pH at which the enteric coatwill dissolve can be controlled by the polymers, combination and ratioof selected polymers, and/or their side groups. For example, dissolutioncharacteristics of the polymer film can be altered by the ratio of freecarboxyl groups to ester groups. Enteric coating layers also containpharmaceutically acceptable plasticizers such as triethyl citrate,dibutyl phthalate, triacetin, polyethylene glycols, polysorbates orother plasticizers. Additives such as dispersants, colorants,anti-adhering and anti-foaming agents may also be included.

The compositions of the invention may be formulated as controlledrelease preparations which may be achieved through the use of a polymerto complex or absorb the proteins of the invention. Appropriate polymersfor controlled release formulations include, for example, polyester,polyamino acids, polyvinyl, pyrrolidone, poly (lactic acid), ethylenevinylacetate, ethylene vinylacetate copolymers, and cellulosederivatives such as methylcellulose. Alternatively, it is possible toentrap the proteins of the invention in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres, andnanoparticles.

The compositions of the present invention can further comprise anadditional therapeutic agent.

According to some embodiments, the therapeutic agent is an antibioticagent. The antibiotic agents include, but are not limited to, β-lactams,aminoglycosides, fluoroquinolones, macrolides, novobiocin, rifampicin,oxazolidinones, fusidic acid, mupirocin, pleuromutilins, daptomycin,vancomycin, tetracyclines, sulfonamides, chloramphenicol, trimetoprim,fosfomycin, cycloserine, polymyxin, azithromycin, clarithromycin,dirithromycin, erythromycin, troleandomycin, roxithromycin, spiramycin,aztreonam, imipenem, meropenem, ertapenem, doripenem,panipenem/betamipron, biapenem, PZ-601, cefixime, cefdinir, cefditoren,cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, cefepime, demeclocycline, doxycycline,minocycline, oxytetracycline, tetracycline, bacitracin, colistin,ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin.

The antibiotic agent can be one or more of the known host-defensepeptides (HDPs).

According to some embodiments, the composition of the invention improvesthe activity of the antibiotic agent. Improving the efficacy of theantibiotic agent includes any aspect of improving or enhancing theeffect of the antibiotic agent, e.g., so that the anti-biofilm effect ofthe antibiotic agent is increased or enhanced in any way over the effectof the antibiotic agent seen in the absence of the mixtures according tothe invention. This may be seen, for example, in a stronger effect ofthe antibiotic agent in inhibiting growth of the bacteria, requiringless antibiotics to achieve the same effect seen in the absence of themixtures according to the invention, or an increased effectiveness seenas increased speed or rate of action, a biofilm eradicating effect beingseen in less time than in the absence of said mixtures.

According to further embodiments, the therapeutic agent is an antiviraldrug or an anti-fungal drug.

Therapeutic Uses of Random Sequence Peptide Mixtures

The present invention provides uses of the pharmaceutical compositionscomprising a therapeutically effective amount of random sequence peptidemixtures of the invention for disrupting biofilm in a subject in need.

According to an aspect, the present invention provides a method fortreating a biofilm-associated infection in a subject comprisingadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of a mixture of random-sequencepeptides according to the invention and a pharmaceutically acceptablecarrier.

Biofilm-forming bacteria, such as staphylococci, Staphylococcus aureus,Enterococcus faecalis, Streptococcus spp., Escherichia coli, Klebsiellapneumoniae, Acinetobacter spp., Proteus mirabilis, Pseudomonas aeruginsaand Candida spp, Staphyloccus saprophyticus, Staphyloccocus xylosus,Staphyloccocus lugdunensis, Staphyloccocus schleiferi, Stapylococcuscaprae, Staphylococcus epidermidis, Staphylococcus hominis,Staphylococcus saprophyticus, Staphylococcus warneri, MRSA, Enterococcusfaecalis (including Vancomycin-resistant enterococcus VRE),Proprionibacterium acnes, Bacillus cereus, Bacillus subtilis, Listeriamonocytogenes, Streptococcus pyrogenes, Streptococcus salivarius, orStreptococcus mutans are associated with a broad spectrum ofbiofilm-associated infections, particularly of nosocomial infections.The bacterial microorganisms form biofilm to be protected against theimmune system. The mixtures of the inventions are useful in killing thebiofilm forming bacteria as well as other bacteria that may be protectedby the biofilm.

The methods of the invention are useful in treating anybiofilm-associated infection. The infection can be, for example, in theoral cavity, the reproductive tract, the urinary tract, the respiratorytract, the gastrointestinal tract, the peritoneum, the middle ear, theprostate, vascular intima, the eye, including the conjunctiva or cornealtissue, in the lung tissue, heart valves, skin, scalp, nails, in wounds;or in the blood.

Typical bacterial infections associated with biofilms in humans are:wound infections, in particular wounds associated with diabetesmellitus, tonsillitis, osteomyelitis, bacterial endocarditis, sinusitis,infections of the cornea, urinary tract infection, infection of thebiliary tract, infectious kidney stones, urethritis, prostatitis,catheter infections, gastrointestinal infections, Legionnaire's disease,middle-ear infections, dental plaques, gingivitis, periodontitis, cysticfibrosis, and infections of permanent indwelling devices such as jointprostheses, and heart valves.

Skin infections include, but are not limited to, cellulitis,folliculitis, impetigo, and boils.

The infection may be acute, or alternatively chronic, e.g., an infectionthat has persisted for at least 5 or at least 10 days, particularly atleast 20 days, more particularly at least 30 days, most particularly atleast 40 days.

The biofilm-associated infection can occur in any subject but somesubjects will be more susceptible to infection than others. Subjects whoare susceptible to these infections include, but are not limited to,subjects whose epithelial and/or endothelial barrier is weakened orcompromised, subjects whose secretion-based defenses to microbialinfection have been abrogated, disrupted, or weakened, and subjects whoare immunocompromised, immunodeficient or immunosuppressed (i.e., asubject in whom any part of the immune response, or an immune activityis reduced or impaired, whether due to disease or clinical interventionor other treatment, or in any way).

The term “therapeutically effective amount” as used herein refers to anamount of the active agent, namely a mixture according to the inventionthat is sufficient to treat, alleviate, and/or inhibit one or moresymptoms of a disease or disorder associated with the formation ofbiofilm in an individual. The therapeutically effective amount will varydepending on the active agent, formulation, the disease and its severityand the age, weight, physical condition and responsiveness of thesubject to be treated.

The dosage and route of administration used in a method of disrupting abiofilm in a subject according to the present invention depends on thespecific disease/site of infection to be treated.

As used herein, the term “treating” means remedial treatment, andencompasses the terms “reducing”, “suppressing”, “ameliorating” and“inhibiting”, which have their commonly understood meaning of decreasingor arresting an infection.

The pharmaceutical composition of the invention may be administered byany suitable administration route, such as by parenteral or by oraladministration route. According to some embodiments, the route ofadministration is via parenteral injection. According to additionalembodiments, the parenteral route of administration is selected from thegroup consisting of subcutaneous, intramuscular, intradermal,intraperitoneal, intravenous, intraarterial, and intrathecal. Thecompositions of the invention can be administered locally.

The pharmaceutical composition of the present invention can beadministered once a week, twice a week, three times a week for a periodof at least two weeks, three weeks, four weeks, 2 months, 3 months, 4months, 5 months, 6 months, or 12 months or any integer in between asrequired so as to disrupt or prevent the formation of biofilm. Eachpossibility represents a separate embodiment of the invention.

The dosage of random sequence peptide mixture administered can rangefrom about 10 ng/kg to about 500 mg/kg of the subject's weight or anyinteger in between. According to some embodiments, the dosage of randomsequence peptide mixtures administered ranges from about 20 ng/kg toabout 20 mg/kg of the subject's weight. According to some embodiments,the dosage of pharmaceutical composition comprising the mixture of theinvention, when administered intravenously, ranges from about 3 mg/kg to10 mg/kg daily for 2-6 weeks.

According to some embodiments, the method of the present inventioncomprises a combination therapy wherein a therapeutic agent formulatedin a separate composition can be administered before, simultaneously, orafter the compositions of the present invention or in alternateschedule.

Other Uses of Random-Sequence Peptide Mixtures

According to an additional aspect, the present invention provides amethod for preventing the formation of biofilm on a medical device,catheter or implant comprising contacting the medical device, catheteror implant with a composition comprising a mixture of random-sequencepeptides according to the invention.

According to some embodiments, the medical device includes, but is notlimited to, a cardiac rhythm management device (CRMD), aneurostimulator, a pulse generator, a drug pump or infusion device, aphysiological monitoring device, and a textured or smooth breastimplant.

Medical devices include, but are not limited to, disposable or permanentor indwelling catheters, (e.g., central venous catheters, dialysiscatheters, long-term tunneled central venous catheters, short-termcentral venous catheters, peripherally inserted central catheters,peripheral venous catheters, pulmonary artery Swan-Ganz catheters,urinary catheters, and peritoneal catheters), long-term urinary devices,tissue bonding urinary devices, vascular grafts, vascular catheterports, wound drain tubes, ventricular catheters, hydrocephalus shunts,heart valves, heart assist devices (e.g., left ventricular assistdevices), pacemaker capsules, pulmonary ventilators, incontinencedevices, penile implants, small or temporary joint replacements, urinarydilator, cannulas, elastomers, surgical instruments, dental instruments,tubings, such as intravenous tubes, breathing tubes, dental water lines,dental drain tubes, feeding tubes, fabrics, paper, adhesives (e.g.,hydrogel adhesives), bandages, orthopedic implants, and any other deviceused in the medical field.

Medical devices also include any device which may be inserted orimplanted into a human or other animal, or placed at the insertion orimplantation site such as the skin near the insertion or implantationsite, and which include at least one surface which is susceptible tocolonization by biofilm embedded microorganisms.

The compositions comprising the mixture of the invention can be used asa sanitizing agent. Said sanitizing agent can be used, for example,before or after surgery.

The composition can be further used as a disinfectant e.g., in humanorgan surgery, in dental surgery, in animal surgery. For that aim, themixtures of the invention can be prepared in a composition in the formof e.g., a spray, a fluid, a powder, a gel, or as an ingredient of a wetwipe or a disinfection sheet product. Said compositions may additionallycomprise suitable carrier, additives, diluting agents and/or excipientsfor its respective use and form.

According to another aspect, the present invention provides an item,such as, a medical device, catheter or implant, coated with or coveredby the composition of the present invention. The coating of thecomposition comprising the mixture of random-sequence peptides can be insome embodiments via covalent binding so that the peptides areimmobilized to the surface of the item.

The surface can be made of any material. For example, it may be metal,e.g., aluminum, steel, stainless steel, chrome, titanium, iron, alloysthereof, and the like. The surface can also be plastic, for example,polyolefin (e.g., polyethylene, (Ultra-High Molecular Weight)polyethylene, polypropylene, polystyrene, poly(meth)acrylate,acrylonitrile, butadiene, ABS, acrylonitrile butadiene, etc.), polyester(e.g., polyethylene terephthalate, etc.), and polyamide (e.g., nylon),combinations thereof, and the like. Other examples include acetalcopolymer, polyphenylsulfone, polysulfone, polythermide, polycarbonate,polyetheretherketone, polyvinylidene fluoride, poly(methyl methacrylate)and poly(tetrafluoroethylene). The surface can also be ceramic,porcelain, gold, and the like.

The compositions comprising a mixture of random-sequence peptidesaccording to the invention can be used for the prevention ofGram-negative and/or Gram-positive bacterial contamination associatedwith bacterial biofilm of food stuff, of food processing equipment, offood processing plants, and of water systems.

Surfaces exposed to microbial contact or contamination include inparticular any part of: food or drink processing, preparation, storageor dispensing machinery or equipment, air conditioning apparatus,industrial machinery, e.g., in chemical or biotechnological processingplants, and storage tanks. Any apparatus or equipment for carrying ortransporting or delivering materials is susceptible to microbialcontamination. Such surfaces will include particularly pipes (which termis used broadly herein to include any conduit or line). Representativeinanimate or abiotic surfaces include, but are not limited to, foodprocessing, storage, dispensing or preparation equipment or surfaces,tanks, conveyors, floors, drains, coolers, freezers, equipment surfaces,walls, valves, belts, pipes, air conditioning conduits, coolingapparatus, food or drink dispensing lines, heat exchangers, boat hullsor any part of a boat's structure that is exposed to water, dentalwaterlines, oil drilling conduits, contact lenses and storage cases.Surfaces of water systems exposed to microbial contact or contaminationinclude in particular any part of: pipes and tubes, valves, filters,reservoirs, basins, and any part in water irrigation systems.

According to some embodiments, the composition can be a cosmeticcomposition. For example, the cosmetic composition can be used foreliminating, reducing and/or preventing biofilm-associated bacterialgrowth on a human skin.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Materials and Methods (i) Chemicals

Fmoc-protected L/D α-amino acids with acid-labile side-chain protectinggroups were purchased from Novabiochem. N-hydroxybenzotriazole (HOBt),N,N-dimethylformamide (DMF), and N,N-diisopropylethylamine (DIEA) werepurchased from Sigma-Aldrich.2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) was purchased from Anaspec. Dehydrated LB culture medium (244610)was obtained from BD (Franklin Lakes, N.J.). All other chemicals werepurchased from Sigma Aldrich and used without purification.

(ii) Synthesis of Random-Sequence Peptide Mixtures

Random peptide mixtures were synthesized using microwave irradiation onRink Amide resin (Substitution 0.2 mmol/gr, 25 μmol) in Alltech filtertubes. Coupling reactions were conducted with binary combinations ofL/D-protected amino acids, with a freshly prepared stock solution thatcontained the protected amino acids in 1:1 molar ration, which was usedfor each coupling step. Before each coupling step, an aliquot containing4 equiv. (100 μmol) of the amino acid mixture was activated with 4equiv. of HBTU, 4 equiv. of HOBt, and 8 equiv. of DIEA, in DMF. Afterthe activated amino acid solution was added to the solid-phase synthesisresin, the reaction mixture was heated to 70° C. in a MARS V multimodemicrowave (2 minute ramp to 70° C., 4 minute hold 70° C.) with stirring.Fmoc deprotection reactions used 20% piperidine in DMF. Reactionsolutions were heated to 80° C. in the microwave (2 minute ramp to 80°C., 2 minute hold 80° C.) with stirring. After eachcoupling/deprotection cycle the resin was washed 3 times with DMF. Uponcompletion of the synthesis, the peptide mixture was cleaved from theresin by stirring the resin in a solution containing 95% trifluoroaceticacid (TFA), 2.5% water, and 2.5% triisopropylsilane for 3 hours. Thepeptide mixture was precipitated from the TFA solution by addition ofcold ether. The precipitated peptide mixture was collected bycentrifugation. Ether was removed, and the pellet was dried under astream of nitrogen, frozen in dry ice and lyophilized.

(iii) Bacterial Growth Inhibition Assays (MIC)

Assays were performed as previously reported (Hayouka, Z. et al., J. AmChem Soc. 2013, 135: 32). The bacteria used in these assays wasmethicillin resistant Staphylococcus aureus 1206 (Weisblum, B. et al., JBacteriol, 1969, 98, 447). Antibacterial activities were determined insterile 96-well plates (BD Falcon 353072 tissue culture plates).Bacterial cells were grown overnight at 37° C. on agar, after which abacterial suspension of approximately 2×10⁶ CFU/mL in Luria Bertani (LB)growth medium was prepared. Samples (50 μL) of this suspension wereadded to 50 μL of medium containing the random-sequence peptide mixturein 2-fold serial dilutions for a total volume of 100 μL in each well.The plates were then incubated at 37° C. for 6 or 24 hours. Bacterialgrowth was determined by measuring the optical density (OD) at 650 nm(Tecan Safire plate reader). The positive control was OD without theaddition of peptide mixture. The minimum inhibitory concentration (MIC)is defined as the lowest concentration at which complete inhibition ofbacterial growth was observed (no increase in OD over the course of theexperiment).

(iv) Biofilm Assay

Clinical isolate strain of S. aureus (1206) was used. Bacterial cellswere grown overnight at 37° C. on agar, after which a bacterialsuspension of approximately 2×10⁶ CFU/mL in LB medium was prepared. 100μL of the bacterial suspension was then inoculated in several wells of a96 well plate and incubated overnight at 37° C. The supernatant liquidwas discarded and the wells were washed with milli-Q water three times.100 μL of LB medium containing the random peptide mixture or antibioticin 2-fold serial dilutions were added to the wells containing thebiofilm and the plates were incubated overnight at 37° C. (in thebiofilm biomass assay for daptomycin, all antibiotic/biofilm mixture atvaried daptomycin concentration contained 1 mM CaCl₂). The supernatantliquid was discarded. The wells were then washed with milli-Q waterthree times.

(v) Crystal Violet Assay for Biomass Quantification

The wells were treated with 125 μL of 0.1% crystal violet and incubatedfor 15 min at 37° C. Excess crystal violet was washed off thoroughlywith milli-Q water three times. 125 μL of 30% acetic acid was added toeach well and the solution was transferred to a new 96-well plate, andabsorbance was measured at 550 nm (Tecan Safire plate reader) using 30%acetic acid in water as blank. The results were expressed as apercentage of biomass in the control biofilm, which was grown withoutany treatment.

(v) XTT Assay for Cell Viability within Biofilm

Cell viability in the biofilms was quantified after incubation withrandom-sequence mixture or antibiotic for 24 hours by an XTT assay. XTT(1 mg/mL) and phenazine methosulfate in the ratio 3:1 were freshlyprepared in 1×PBS buffer. Following the washing step of the biofilmafter treatment, 100 μL of this solution was added to the wells and theplates were incubated at 37° C. for 3 hours in the dark. An aliquot of90 μL was taken and transferred to a 96-well plate and OD was measuredat 490 nm (Tecan Safire plate reader) using the XTT solution as blank.The results were expressed as a percentage of cell viability relative tothe control biofilm without any treatment.

Example 1 Effect of Random Sequence Peptide Mixtures on the Growth ofMethicillin-Resistant Staphylococcus aureus

The ability of the random-sequence peptide mixture to inhibit the growthof planktonic MRSA cells after 6 h or 24 h was examined. As shown inFIG. 1, 20-mer random-sequence peptide mixtures of 1:1 homochiral orheterochiral phenylalanine-lysine (^(L)F^(L)K or ^(L)F^(D)K) inhibitedthe growth of planktonic MRSA at a very low concentration. The MinimalInhibition Concentration (MIC) was 6 μg/mL for 6 h incubation and 12μg/mL for 24 h incubation.

Example 2 Random Sequence Peptide Mixtures Disrupt Biofilms

Biofilm biomass disruption was quantified using an assay that measurescrystal violet absorbance at 550 nm to assess surface-attached biomass(O'Toole, G. A. et al., Mol Microbiol 1998, 30, 295-304). The resultsindicated that heterochiral mixture of 1:1 phenylalanine and lysine(^(L)F^(D)K) at 25 μg/mL showed stronger biofilm biomass disruption (22%of the biofilm biomass remained) compared to the homochiral (^(L)F^(L)K)mixture (55%). At a higher concentration (200 μg/mL), bothrandom-sequence peptide mixtures (homochiral and heterochiral) werehighly active (FIG. 2). The differences in the observed activity profileare probably due to the stability of the mixtures. The homochiralpeptide mixture is probably more sensitive to protease activity than theheterochiral peptide mixture. Daptomycin, a lipopeptide antibiotic thatis being used in the treatment of systemic and life-threateninginfections caused by MRSA was used as a positive control in this study.Daptomycin was shown previously to disrupt biomass biofilm of MRSA.Biofilm biomass disruption was also evaluated for a mixture of 1:1leucine and lysine. As shown in FIG. 2, lower activity was observed forthe leucine-lysine mixture.

Next, XTT assay was used to quantify the cell viability of bacterialcells in the biofilm matrix after treating with the random-sequencepeptides mixtures for 24 hr. The results showed that only 5% of the MRSAbacterial cells survived the treatment of the heterochiral mixture,compared to a significantly higher extent of cell survival (40%) whentreated with the homochiral mixture (FIG. 3). The killing of thebacterial cell within the biofilm was further evaluated by cellcounting. The biofilm biomass was sonicated and plated to measure theviability of MRSA inside the biofilm biomass after treatment. Theheterochiral mixture treatment killed most of the bacterial cells withinthe biofilm biomass (FIG. 4).

For further characterization of the ability of the mixtures to eradicatethe MRSA established biofilm, a Scanning Electron Microscopy (SEM) studywas performed. A mature MRSA biofilm was grown on glass disks for 48 h,then was treated with 100 μg/mL of ^(L)F^(L)K or ^(L)F^(D)K for 24 h andviewed under a scanning electron microscope (FIGS. 5A, 5B and SC). Asshown in the figure, ^(L)F^(D)K was able to remove MRSA biofilms moreefficiently (FIG. 5C) compared to the homochiral mixture ^(L)F^(L)K(FIG. 5B).

Example 3 Effect of Random Sequence Peptide Mixtures of Tryptophan andLysine on B. subtilis Forming Biofilm

The effect of random sequence peptide mixture on biofilm formation wasinvestigated using mixtures of peptides prepared with tryptophan andlysine. The anti-biofilm effect of random-sequence peptide mixtures thatcontain tryptophan and lysin in 4 different stereoisomeric forms(^(L)W^(L)K, ^(L)W^(D)K, ^(D)W^(L)K, ^(D)W^(D)K) was examined on B.subtilis 3610.

The Bacillus subtilis wild strain NCIB3610 and Bacillus subtilis thatwas isolated from milk were used in this study. Lactobacillus-MRS Broath(LMRS Broath) is an enriched selective medium intended for the isolationand cultivation of Lactobacillus found in clinical specimens and dairyand food products. This media was found to be very effective to growBacillus subtilis pellicle, a biofilm floating at the air-liquidinterface. For biofilm generation, bacteria were grown to stationaryphase in LMRS at 37° C. in shaking culture to around 1×10⁸ CFU per ml.Biofilms were generated at 30° C. in the biofilm promoting medium LMRS.As shown in FIG. 6A, a very thick biofilm was developed in the controlbacteria (B. sub 3610 lane). In contrary, inhibition in the formation ofbiofilm (50 μg/mL) was observed in wells that were treated with WK,W^(D)K, or ^(D)WK random peptide mixtures (FIGS. 6A and 6B). The^(D)W^(D)K peptide mixture exhibited lower activity, wherein biofilmformation was inhibited at 100 μg/mL (FIG. 6B). These resultsdemonstrate that the random peptide mixtures inhibit biofilm formationof B. subtilis.

Example 4 Random-Sequence Peptide Mixtures Penetrate MRSA Cells

To study if the mixtures of the invention penetrate the bacterial cells,random sequence peptide mixtures (^(L)F^(L)K or ^(L)F^(D)K) weresynthesized and labeled at their N′ terminus free amino group with5(6)-carboxyfluorescein. Confocal microscopy was used to determine theirability to penetrate MRSA bacterial cells (FIGS. 7A, 7B and 7C). After 1h of incubation, the heterochiral and homochiral mixtures were observedin MRSA bacterial cells (FIGS. 7B and 7C, respectively). It is assumedthat some of the random peptide mixtures caused damage to the bacterialmembrane, and enhanced the entry of other random peptides into thebacterial cells.

Example 5 Random-Sequence Peptide Mixtures Effect on Human Cells

The random peptide mixtures (^(L)F^(L)K or ^(L)F^(D)K) were examined fortheir red blood cell hemolysis activity as described before (Hayouka etal., J. Am. Chem. Soc., 2013, 135, 11748-11751). The heterochiralpeptide mixture showed lower hemolytic activity towards human red bloodcells compared to the homochiral mixture (FIG. 8).

The cytotoxicity of the random-sequence peptide mixtures was furthertested on Caco-2 (European Collection of Animal cell Cultures, UK)cells. This human intestinal epithelial cell line was used betweenpassages 30-50. Caco-2 cells were grown in Dulbecco's modified Eagle'smedium (DMEM) with 2 mM L-glutamine, 1% (v/v) non-essential amino acids,100 U/ml penicillin, 100 μg/ml streptomycin and 10% (v/v) fetal bovineserum (Sigma-Aldrich, Ireland). The cells were grown at 37° C. in ahumidified atmosphere with 5% CO₂. The cytotoxic potential of therandom-sequence peptide mixtures was determined following incubation ofexponentially growing cells using the MTT assay. As shown in FIG. 9,random-sequence peptide mixtures (^(L)F^(L)K or ^(L)F^(D)K) exhibitedless cytotoxicity compared to leucine-lysine mixture at 200 and 400μg/ml concentrations.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

What is claimed is:
 1. An item configured to prevent biofilm formation,disrupt biofilm and/or prevent microbial contamination, wherein the itemincludes a coating or covering of a composition comprising a mixture ofa plurality of random-sequence peptides, wherein the peptides have anidentical number of amino acid residues and a length of 10 to 40 aminoacid residues, wherein the mixture of peptides consists of one or morespecies of hydrophobic amino acids in an L- or D-configuration and oneor more species of cationic amino acids in an L- or D-configuration,wherein the ratio in the mixture of the total hydrophobic and cationicamino acids is about 3:1 to 1:3, and wherein the mixture of theplurality of random-sequence peptides contains 2¹⁰ up to millions ofsequences.
 2. The item of claim 1, wherein the biofilm to be preventedor disrupted or the microbial contamination to be prevented is due tounicellular organisms selected from the group consisting of bacteria,fungi, and archaea.
 3. The item of claim 2, wherein the bacteria areselected from the group consisting of Staphylococcus aureus,Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus,Streptococcus mutans, E. coli, Listeria, Salmonella, Klebsiella,Enterococcus, and Pseudomonas aeruginosa.
 4. The item of claim 2,wherein the bacteria are antibiotic-resistant bacteria.
 5. The item ofclaim 1, wherein the hydrophobic amino acid is selected from the groupconsisting of phenylalanine, leucine, tryptophan, and combinationsthereof, and wherein the cationic amino acid is lysine.
 6. The item ofclaim 5, wherein the mixture consists of phenylalanine and lysine, orleucine and lysine, or tryptophan and lysine.
 7. The item of claim 6,wherein the mixture consists of leucine and lysine and the ratio ofleucine and lysine in the mixture is about 1:1 to 1:3.
 8. The item ofclaim 1, wherein the peptides have a length of 15 to 25 amino acids. 9.The item of claim 1, wherein the composition is coated by immobilizingthe peptides to the surface of the item.
 10. The item of claim 1selected from the group consisting of a medical device, a food or drinkpackaging, a food or drink container, food, food or drink processingequipment, food or drink preparation equipment, and water systems or anypart thereof.
 11. A method for preventing biofilm formation, disruptinga biofilm and/or preventing microbial contamination comprising applyingto a surface of an item a composition comprising a mixture of aplurality of random-sequence peptides, wherein the peptides have anidentical number of amino acid residues and a length of 10 to 40 aminoacid residues, wherein the mixture of peptides consists of one or morespecies of hydrophobic amino acids in an L- or D-configuration and oneor more species of cationic amino acids in an L- or D-configuration,wherein the ratio in the mixture of the total hydrophobic and cationicamino acids is about 3:1 to 1:3, and wherein the mixture of theplurality of random-sequence peptides contains 2¹⁰ up to millions ofsequences.
 12. The method of claim 11, wherein the biofilm to beprevented or disrupted or the microbial contamination to be prevented isdue to unicellular organisms selected from the group consisting ofbacteria, fungi, and archaea.
 13. The method of claim 12, wherein thebacteria are selected from the group consisting of Staphylococcusaureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus,Streptococcus mutans, E coli, Listeria, Salmonella, Klebsiella,Enterococcus, and Pseudomonas aeruginosa.
 14. The method according toclaim 12, wherein the bacteria are antibiotic-resistant bacteria. 15.The method according to claim 11, wherein the hydrophobic amino acid isselected from the group consisting of phenylalanine, leucine,tryptophan, and combinations thereof, and wherein the cationic aminoacid is lysine.
 16. The method of claim 15, wherein the mixture consistsof phenylalanine and lysine, or leucine and lysine, or tryptophan andlysine.
 17. The method of claim 16, wherein the mixture consists ofleucine and lysine and the ratio of leucine and lysine in the mixture isabout 1:1 to 1:3.
 18. The method of claim 11, wherein applying thecomposition comprises applying the composition as a coating or coveringof the item or otherwise immobilizing the peptides to the item'ssurface.
 19. The method of claim 11, wherein the item is selected fromthe group consisting of a medical device, a food or drink packaging, afood or drink container, and water systems or any part thereof.
 20. Themethod of claim 11 useful for food or drink processing and/or food ordrink preparation.